An investigation of the temporal induction of cytokine mRNAs in LPS-challenged thioglycollate-elicited murine peritoneal macrophages using the reverse transcription polymerase chain reaction

• Published in: Inflammation research Vol. 46; no. 2; pp. 65 - 71
• Main Authors: Simpson, A. E. C. M., Tomkins, P. T., Cooper, K. L.
• Format: Journal Article
• Language: English
• Published: Switzerland 01.02.1997
• Subjects: Animals; Cytokines - biosynthesis; Cytokines - genetics; Female; Interleukin-1 - biosynthesis; Interleukin-1 - genetics; Interleukin-10 - biosynthesis; Interleukin-10 - genetics; Interleukin-12 - biosynthesis; Interleukin-12 - genetics; Interleukin-6 - biosynthesis; Interleukin-6 - genetics; Lipopolysaccharides - pharmacology; Macrophages, Peritoneal - drug effects; Macrophages, Peritoneal - metabolism; Mice; Mice, Inbred BALB C; Polymerase Chain Reaction; RNA, Messenger - biosynthesis; RNA, Messenger - chemistry; RNA, Messenger - metabolism; Thioglycolates - metabolism; Thioglycolates - pharmacology; Tumor Necrosis Factor-alpha - biosynthesis; Tumor Necrosis Factor-alpha - genetics
• ISSN: 1023-3830; 1420-908X
• DOI: 10.1007/s000110050078

A comprehensive study to standardise interleukin (IL)-1 alpha, -1 beta, -6, -10, -12 and tumour necrosis factor alpha (TNF alpha) mRNA detection in murine peritoneal macrophages, using the reverse transcription polymerase chain reaction (RT-PCR) was carried out. Thioglycollate-elicited peritoneal exudate cells were harvested from female BALB/c mice and the adherent macrophage fraction isolated for use. Peritoneal macrophages (1 x 10(6)) were incubated in the presence or absence of lipopolysaccharide (LPS; at a final concentration of 1 microgram/ml) for 0, 1, 2, 3, 4, 5 and 24 h. Culture supernatants and cells were harvested at each time point, the secreted cytokine protein levels quantified by sandwich immunoassays and the cytokine mRNA levels assessed by RT-PCR. The IL-6 mRNA was not expressed in detectable amounts in the macrophages, unless challenged with LPS. TNF alpha, IL-1 alpha, IL-1 beta, IL-10 and IL-12 mRNAs were expressed in both stimulated and unstimulated macrophages. The levels of the PCR products and thus mRNAs of all the cytokines increased with LPS stimulation, maximal levels being achieved 3 to 5 h post stimulation. RT-PCR produced consistent results, indicating that this technique could be used to investigate the effect of biological mediators and novel pharmacological agents on cytokine mRNA levels in macrophages.